The prospective effect of fucoidan on splenic dysfunction caused by oxaliplatin in male rats through endoplasmic stress dynamics

Fucoidans (FUCs) are highly sulfated polysaccharides demonstrating multiple actions in different systems. Oxaliplatin (OXA) is a platinum-containing chemotherapeutic agent with several side effects that restrict its usage. The current study aimed to determine the potential effect of FUC in male rats with splenic dysfunction induced by OXA. Eighty adult male rats aged (8–9 weeks) weighing (190–230 g) were divided into four groups: (Group I: the control group): Rats were administrated normal saline; (Group II: controls treated by FUC): Rats were treated with FUC; (Group III: Splenic dysfunction group): Rats were treated with 8 mg/kg OXA. (IV: Splenic dysfunction treated by FUC): Rats were treated by OXA as Group III, then fucoidan was given. At the end of the experiment, blood was collected to determine red blood cells and white blood cells. Splenic tissues were divided into one part for biochemical assays, oxidative stress markers as MDA and catalase, inflammatory markers (TNF-alpha, IL6), and apoptotic markers (caspase 3) and gene expression of Nrf2, Mapk1 gene expression, and endoplasmic stress parameters and the other part was used for immunohistochemical and histopathological analysis. Compared to the OXA-induced splenic dysfunction group, FUC significantly decreased high levels of MDA, TNF- alpha, IL6, caspase-3, Mapk1, endoplasmic stress induced by OXA, and increased the level of catalase and Nrf2. Fucoidan has corrected the histopathological and immunohistochemical changes compared to the OXA-induced splenic dysfunction group. In conclusion, our findings suggest that fucoidan has a significant role in the treatment of splenic dysfunction induced by OXA.

Real-time PCR. Total RNA was extracted from frozen splenic tissue after processing using Qiagen RNeasy Total RNA isolation kit (Qiagen, Hiden, Germany) according to the protocol provided by the manufacturer. By a NanoDrop spectrophotometer (NanoDrop Technologies, Inc. Wilmington, USA), The total RNA concentration and purity were measured at the OD260 and OD260/280 ratios, respectively. RNA has an A260/A280 ratio of 1.9-2.2 and the RNA was then preserved at-80° C. This was followed by synthesis of the first strand using Super-Script III First-Strand Synthesis System for real-time PCR kit (Life Technologies, Carlsbad, California, USA) according to manufacturer's instructions. PCR reactions were performed using Power SYBR Green PCR Master Mix (Life Technologies, Carlsbad, California, USA) following the manufacturer's instructions. MAPK, Nrf2, GRP78, CHOP and DPP4 mRNA transcripts were quantified relative to the housekeeping gene GAPDH gene, which was used as an internal control. Sequence specific primers were designed ( Table 1). The thermal cycling conditions were as follows: Initial denaturation at 95 °C for 10 min was followed by 40 cycles with denaturation at 95 °C for 15 s, annealing at 60 °C for 30 s and extension at 72 °C for 30 s. At the end of the last cycle, the temperature was increased from 60 to 95 °C for melting curve analysis. Relative gene expression was automatically calculated using the comparative threshold (Ct) method for the values of the target and the reference genes using the 2 −ΔΔCT formula.
Histopathological evaluation of splenic tissues. Splenic samples were fixed in 10% formalin and embedded in paraffin. Five µm sections of formalin fixed paraffin embedded (FFPE) blocks were stained with hematoxylin and eosin (H&E) for routine histopathological evaluation. Slides were examined for histopathological changes such as architectural abnormality, inflammatory response, vascular congestion, and apoptosis. Immunohistochemical analysis. Tissue sections were deparaffinized in xylene and dehydrated with graded concentrations of alcohol. Antigen retrieval was done by immersion in citrate buffer (pH 6.0) for 10 min at 95 °C. Then the tissue samples were naturally incubated with 0.3% H 2 O 2 for 15 min to block the endogenous peroxidase activity.
After washing in phosphate-buffered saline (PBS), incubation with anti-bcl-2 antibodies (C-2: sc-7382, Santa Cruz Biotechnology, INC, USA, dilution 1:100) was done, for 2 h at room temperature. Slides were then washed and incubated biotinylated secondary antibodies for 20 min. After washing, sections were incubated with diaminobenzidine substrate-chromogen solution (DAB) for 30 s. Finally, all sections were counterstained with Mayer's hematoxylin. Negative controls were performed by replacing the primary antibody with PBS. Assessment of bcl-2 immunohistochemical staining. Cytoplasmic staining was interpreted as positive for bcl-2. Bcl-2 scoring was as follows, considering ten high power fields (400×): score 0 (no staining), score + 1 (weak positive, staining of < 10% of the cells), score + 2 (moderate, staining of 10-75% of the cells), and score + 3 (strong positive, staining of > 75% of the cells). Scores of 0 and + 1 were considered negative, and scores of + 2 and + 3 were considered positive for bcl-2 expression 16 .
Morphometric study. Five non-overlapping random fields (magnification: 400×; area: 0.071 mm 2 ) of splenic tissue sections were chosen, photographed, and submitted for morphometric study. Quantification of optical color density and the mean area percentage of bcl2 immunohistochemical positive cells in DAB-stained sections was performed using Image software (Media Cybernetics).

Effect of FUC on oxidant/antioxidant biomarkers, apoptosis and inflammatory biomarkers.
The splenic catalase level was decreased significantly in OXA-treated group in comparison to other studied groups (P < 0.05) while MDA and NO levels showed the reverse. FUC co-treatment significantly increased catalase level and significantly decreased MDA and NO levels (P < 0.05), Inflammatory biomarkers TNF-α and IL-6 levels showed a significant increase in OXA treated group when compared to other studied groups (P < 0.05).
On the other hand, FUC co-treatment significantly decreased all the aforementioned inflammatory biomarkers (P < 0.05),splenic tissue caspase 3 level showed a significant increase in OXA treated group when compared to other studied groups (P < 0.05). On the other hand, FUC co-treatment significantly decreased caspase 3 level in splenic tissue (P < 0.05) ( Table 2).

Effect of FUC on some hematological parameters (RBCs and WBCs counts). RBCs and WBCs
counts showed a significant decrease (anemia and leucopenia, respectively) in OXA treated group when compared to other studied groups (P < 0.05). On the other hand, FUC co-treatment significantly increased RBCs and WBCs counts (P < 0.05) ( Histopathological results. Splenic tissue in the control group showed normal architecture with normal red pulp and white pulp with central arteriole. Also, the control treated by FUC group showed normal architecture of splenic tissue. Splenic tissue in the group III (Splenic dysfunction) showed disturbed architecture in the form of irregular white pulp and congested red pulp. Its higher magnification showed red pulp with extramedullary hematopoiesis with many megakaryocytes and apoptotic bodies. Splenic tissue in the group IV (Splenic Table 2. Effect of FUC on oxidant/antioxidant biomarkers, apoptosis and inflammatory biomarkers among the studied groups. Data are represented as mean ± SD. Statistical analysis was carried out using one-way ANOVA with Tukey's post hoc test, SPSS computer program. a-d Significant difference between groups at *p < 0.05. a Significance from group I; b significance from group II; c significance from group III; d significance from group IV. Degree of freedom is (3) between groups and (24) within groups (total = 27). TNF-α Tumor necrosis factor-alpha, IL-6 Interleukin-6, MDA MDA, NO Nitric oxide.  www.nature.com/scientificreports/ dysfunction treated by FUC) showed preserved architecture with clearly defined red pulp and white pulp with central arteriole (Fig. 6).
Immunohistochemical and morphometric results. Bcl-2 expression in splenic tissue in the control group showed positive expression of the red pulp as well as the mantle zone of the white pulp while the germinal center of the white pulp showed negative bcl-2 expression. Also, Bcl-2 expression in the control treated by FUC group showed positive expression of the red pulp. Bcl-2 expression in the group III (Splenic dysfunction) showed negative expression of the red pulp. Bcl-2 expression in the group IV (Splenic dysfunction treated by FUC) showed positive expression of the red pulp ( Fig. 7a-d). There was a significant decrease in Bcl-2 color density and the mean area percentage in group III (Splenic dysfunction) (0.23 ± 0.036, 4.68 ± 2.34) respectively compared to the control group (0.75 ± 0.17, 65.38 ± 3.5 respectively (p < 0.05). It increased significantly to normal levels in group IV (0.64 ± 0.089, 55.61 ± 4.15) respectively compared to group III (p < 0.05) (Fig. 7e,f).

Discussion
The current study demonstrated that 8-week FUC treatment significantly treats splenic dysfunction caused by OXA in male rats as shown by our biochemical, histopathological, and immune-histochemical results. www.nature.com/scientificreports/ Our results showed that rats of OXA-induced splenic dysfunction group showed significantly increased OS which is evidenced by the significant increase of MDA and NO level and significant decrease of catalase.
Excessive levels of reactive oxygen species (ROS) damages the cells directly by eliciting lipid peroxidation, protein degradation with subsequent DNA damage. Notably, whenever the ROS production exceeds the ability of the cell's natural antioxidant defenses to sequester, OS develops 17 .
The OXA-induced OS is related to several factors, including the fact that OXA causes mitochondrial dysfunction by damaging mitochondrial DNA and interrupting RNA expression of Cytochrome B, resulting in disruption of mitochondrial function necessitating antioxidant therapy 18 . www.nature.com/scientificreports/ Furthermore, mitochondrial damage activates nitric oxide synthase (NOS), and NO's direct toxicity is significantly increased through the reaction with superoxide and eventually yielding into peroxynitrite, that in turn changes protein's tyrosines into nitrotyrosines, resulting in severe OS 9 .
Our results also showed that OXA-treated splenic tissue exhibit significantly decreased expression NRF2 also indicating OS. NRF2, a central transcription factor, plays a crucial role in producing antioxidant and detoxifying enzymes. The physiological binding of Nrf2 to Ketch-like ECH-associated protein (Keap1) is disrupted by OS with subsequent upregulation of the transcription of antioxidant response element (ARE)-controlled genes. This in turn upregulates the expression of a number of antioxidant and phase II drug metabolizing enzymes, such as HO-1 and NQO1, which inhibit OS 19 .
Another factor contributing to the OXA-induced splenic dysfunction was found to be the increased splenic cell apoptosis as indicated by the significant increase of caspase3 and significant decrease of BCL-2, demonstrated in the current study, together with the previously reported upregulated expression of apoptosis-related proteins, such as Bax. The OXA-induced activation of both inflammatory and apoptotic pathways was also evidenced in the current study by the significantly increased splenic Mapk1 protein expression.Bcl-2, a member of the Bcl-2 protein superfamily, is found on the outer mitochondrial membrane preventing the mitochondrial liberation of cytochrome C and other pro-apoptotic molecules into the cytosol 20 .
On the other hand, the extracellular signal-regulated kinase, c-Jun N-terminal kinase (JNK), and p38 Mapk1 pathways are all members of the Mapk1 family. The Mapk1 signaling pathway induces p38 phosphorylation, activates transcription factors eventually speeding up the apoptotic process indicating the ROS-induced upregulation of the JNK and p38 MAPK pathways 12 .
Previous studies explained that the OXA-induced apoptosis is attributed to the increased ROS production, Nox1-associated with JNK/p38-MAPK activation and survivin degradation by p38 activation 21 . www.nature.com/scientificreports/ This causes many proapoptotic events, including p53 activation, Bax translocation, cytochrome c release, and activation of caspase-3 and 9. Also, OXA upregulates the calcium N channel which increases the intracellular calcium accumulation aiding the apoptotic mechanisms 18 .
Moreover, After OXA therapy, the antiapoptotic protein Bcl-2 level is significantly decreased, Bcl-2 is a member of the Bcl-2 protein superfamily, which includes antiapoptotic proteins. Bcl-2 is found on the outer mitochondrial membrane and prevents the release of pro-apoptotic molecules from the mitochondria to the cytosol such as cytochrome C 15 .
Another suggested mechanism for OXA-induced apoptosis is that it is capable of inhibiting tumor-associated NADH oxidase (tNOX) thus, decreasing the cell's NAD+/NADH ratio and inhibiting the SIRT1 deacetylase activity via the tNOX-induced modulation of the NAD+-SIRT1 axis that results in apoptosis 22 .
The current study reported OXA-induced inflammatory changes in splenic tissue as indicated by the significant increase of TNF-α and IL-6. The developed inflammatory process contributed to the splenic tissue damage and apoptosis as aforementioned. In agreement with our results 17 , reported the markedly increased splenic TNF-α, IFN-γ and IL-17 in OXA-treated mice with subsequent potent cytotoxic immune response, apoptosis and splenic injury.
The complex interdependent relationship between inflammatory, OS and apoptosis triggered by OXA treatment can be furtherly explained as follows. TNF-α activates caspase-3, which causes necrosis and triggers the apoptotic pathway. OS stimulates many inflammatory pathways, such as nuclear factor (NF-κB) and NLR family pyrin domain containing 3 (nlrp3), with subsequent upregulated expression of inflammatory cytokines. Moreover, the aforementioned ROS-induced triggering of the JNK and caspase pathways results also in apoptosis induced by TNF-α 17 .
Furthermore, our results revealed an OXA-associated ER stress, which is evidenced by the significantly increased expression of protein levels of GRP78, CHOP, and DPP4 in the splenic tissue. These three proteins, also known as the unfolded protein response (UPR) or ER stress response, are the mediators of the signaling cascade responsible for increasing the protein folding capacity in response to ER stress as a method of restoration of the cell's homeostasis 23 . Moreover, ROS excess is also linked to ER stress as reported by Ref. 24 , which implies that OXA treatment with apoptosis is a common sequence to ER stress and activation of the CHOP pathway 23 .
Interestingly, besides splenic dysfunction, OXA treatment induced a significant decrease in RBCs and WBCs, as demonstrated in the current study. Also, in vitro toxicity of OXA was reported in the form of the emergence of polychromatophilic reticulocytes or malformed RBCs, such as spherocytes which were attributed to OXA's ability to bind with hemoglobin and cytokine secreted during cellular stress indicating DNA damage 10 .
The effect of OXA on splenic tissue is evident in animals and humans, and the histopathological results of the current study agree with other previous reports. Histopathological examination of the OXA group showed disturbed architecture and apoptotic bodies, so splenic dysfunction. These changes reflect the chemical changes detected in splenic tissue homogenate.
The results of the current study showed that FUC could alleviate OS, indicated by the significant decrease of MDA and significant increase of catalase. The significant upregulation of splenic NRF2 protein expression also evidences FUC's ameliorative effect on OS.
According to previous studies, FUC is a powerful antioxidant and radical scavenger. The relation between the content of sulfate and radical capacity in scavenging superoxides was positive, and the ratio of sulfate to FUC activity was an effective indicator 5 .
Those reports were supported by the work done by Ref. 25 , which denoted that the FUC active sulfate group enhances the antioxidant ability of FUC that, in turn, can stop Fenton's reaction and ROS generation by chelating transition metal ions necessary for a free radical chain reaction in addition to metal complexes formation.
In a further analysis, the neutralization of NO free radical at 1 mg/ml FUC was fully shown in S. polycystum, which indicates that FUC has a high NO scavenging ability 5 .
These mechanisms are in a parallel line with 26 , who added that FUC inhibited LPO, decreased NO level, and increased the antioxidants near to normal values.
There is no contradiction between these results and the report by Ref. 1 that FUCs help scavenges ROS such as hydroxyl, peroxyl, and superoxide radicals and enhance SOD, catalase, and glucose6 phosphate dehydrogenase (G6PD).
In the present study, FUC induced significantly upregulated expression of Nrf2. FUC-induced increased GSK-3β phosphorylation provides an explanation for these results, especially since glycogen synthase kinase-3β (GSK-3β) is the upstream molecular of the Nrf2 signaling pathway 27 .
FUC also showed a potent anti-inflammatory activity indicated by the significant decrease in TNF-α and IL-6 in splenic tissue. Furtherly explaining, FUC was reported to inhibit leukocytic recruitment, block L-selectin, and hinder the cell-cell interactions, which is evident in our results as well as the results by Ref. 28 in the form of absent inflammatory cell infiltration in comparison with the intoxicated rats as confirmed by results with 26 .
In accordance with its aforementioned anti-inflammatory effect, the current study demonstrated FUCinduced downregulated expression of NFκB, which is confirmed by the findings of Ref. 1 regarding the downregulation of NFκB, protein kinase B, extracellular signal-regulated kinase, c-Jun N-terminal kinase, and p38 mitogen-activated protein kinase expression. In addition, it reduced LPS-enhanced elevation of serum levels of TNF-α, IL-1β, and IL-6 in mice, and it reclines Seth's induced PGE2 and IL-6 plasma levels caused by aspirin.
Moreover, our results also demonstrated that FUC-induced downregulated expression of splenic MAPKs. This finding can be explained by the inhibition of ROS production by FUC, which are an important trigger for Mapks activation 29 .
Interestingly, our work also demonstrated that FUC is an antiapoptotic, evidenced by the decreased caspase 3 level and increased expression of Bcl-2, which was confirmed by the immunohistochemical staining. These results www.nature.com/scientificreports/ are in accordance with the work done by Ref. 30 , who demonstrated that FUC decreased hepatocyte apoptosis with up-regulation of p42/44 Mapk dependent NDRG-1/CAP43 and VMP-1.

Scientific
In addition, FUC inhibited intrinsic and extrinsic apoptosis mediated by the TRADD/TRAF2 and JAK2/ STAT1 pathways that are induced by TNF-α and IFN-γ. These reports represented methods to protect against splenic apoptosis and dysfunction 31 .
Furthermore, FUC treatment induced significant downregulation of expression of protein levels of GRP78, CHOP, and DPP4 in the splenic tissue evidencing improvement of the ER stress. The decrease in ER stress can be attributed to FUC's efficient inhibition of the MAPKs activation, namely (p-JNK and p-P38), as evidenced in our data as well as the report by Ref. 29 .
Other data suggested that FUC protects splenic dysfunction through the activation of AKT and Mapk, with subsequently improved ER stress 29 .
Furthermore, the present study showed that FUC significantly increased WBCs and RBCs count, which supports the previous findings reporting that FUC is a stimulator of hematopoiesis 32 .
These results were parallel with the previous reports that include many mechanisms, including mobilizing leucocytes from bone marrow to peripheral blood, enhanced differentiation of the mobilized CD34+ cells into leucocytes, and the antibacterial and antiviral properties of FUC 33 .
Eventually, FUC treatment successfully ameliorated these pathological effects, as evidenced by showing normal architecture with normal red pulp (blue arrow) and white pulp of splenic tissue in the FUC treated group, which confirms the aforementioned improved splenic tissue parameters.

Conclusion
We concluded that FUC could protect against splenic dysfunction caused by OXA by targeting endoplasmic stress dynamics and improving OXA-induced oxidative, inflammatory, and apoptotic stress in splenic tissue.

Recommendations.
Our data suggest that FUC can be used as a promising adjuvant therapy to ameliorate splenic dysfunction caused by OXA. However, further research is required to fully understand its safety limits, toxicity, and effects on splenic tissue.
The limitations and strengths of this study. Our data suggest that fucoidan can be used as a promising prophylactic therapy to ameliorate splenic damage with OXA administration. However, further research is required to understand its safety limits fully and toxicity, study its effect as a treatment rather than prophylaxis of the toxic effect of OXA on the spleen and verify the mechanism of action of its preservative effect on the leucocytic and RBCs count. In addition, the mechanism of the effect of fucoidan on apoptosis needs further investigation to determine if it is directly or indirectly mediated through its effect on ER dynamics.

Data availability
All data that support the finding of the current study are available from the corresponding author upon reasonable request. Data sharing applies to this article as new data were analyzed in this study.